1. Inhibition of mTOR by Rapamycin Abolishes Cognitive
Deficits and Reduces Amyloid-b Levels in a Mouse Model
of Alzheimer’s Disease
Patricia Spilman8
, Natalia Podlutskaya1,2
, Matthew J. Hart5
, Jayanta Debnath7
, Olivia Gorostiza8
, Dale
Bredesen8
, Arlan Richardson2,4,6
, Randy Strong2,3,6
, Veronica Galvan1,2
*
1 Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America, 2 The Barshop Institute for Longevity
and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America, 3 Department of Pharmacology, University of
Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America, 4 Department of Cellular and Structural Biology, University of Texas Health
Science Center at San Antonio, San Antonio, Texas, United States of America, 5 Department of Molecular Medicine, University of Texas Health Science Center at San
Antonio, San Antonio, Texas, United States of America, 6 Geriatric Research, Education and Clinical Center and Research Service, South Texas Veterans Health Care System,
San Antonio, Texas, United States of America, 7 Department of Pathology, University of California San Francisco, San Francisco, California, United States of America, 8 The
Buck Institute for Age Research, Novato, California, United States of America
Abstract
Background: Reduced TOR signaling has been shown to significantly increase lifespan in a variety of organisms [1,2,3,4]. It
was recently demonstrated that long-term treatment with rapamycin, an inhibitor of the mTOR pathway[5], or ablation of
the mTOR target p70S6K[6] extends lifespan in mice, possibly by delaying aging. Whether inhibition of the mTOR pathway
would delay or prevent age-associated disease such as AD remained to be determined.
Methodology/Principal Findings: We used rapamycin administration and behavioral tools in a mouse model of AD as well
as standard biochemical and immunohistochemical measures in brain tissue to provide answers for this question. Here we
show that long-term inhibition of mTOR by rapamycin prevented AD-like cognitive deficits and lowered levels of Ab42, a
major toxic species in AD[7], in the PDAPP transgenic mouse model. These data indicate that inhibition of the mTOR
pathway can reduce Ab42 levels in vivo and block or delay AD in mice. As expected from the inhibition of mTOR, autophagy
was increased in neurons of rapamycin-treated transgenic, but not in non-transgenic, PDAPP mice, suggesting that the
reduction in Ab and the improvement in cognitive function are due in part to increased autophagy, possibly as a response
to high levels of Ab.
Conclusions/Significance: Our data suggest that inhibition of mTOR by rapamycin, an intervention that extends lifespan in
mice, can slow or block AD progression in a transgenic mouse model of the disease. Rapamycin, already used in clinical
settings, may be a potentially effective therapeutic agent for the treatment of AD.
Citation: Spilman P, Podlutskaya N, Hart MJ, Debnath J, Gorostiza O, et al. (2010) Inhibition of mTOR by Rapamycin Abolishes Cognitive Deficits and Reduces
Amyloid-b Levels in a Mouse Model of Alzheimer’s Disease. PLoS ONE 5(4): e9979. doi:10.1371/journal.pone.0009979
Editor: Pier Francesco Ferrari, Universita` di Parma, Italy
Received December 23, 2009; Accepted March 9, 2010; Published April 1, 2010
Copyright: ß 2010 Spilman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIRG-DDC-120433 from the Alzheimer’s Association to V.G. and by the NIA Interventions Testing Center (U01AG022307) to
R.S. The authors also recognize the support of the San Antonio Nathan Shock Aging Center (P30AG-13319, A.R.) and the VA Neurodegeneration Research Center
(REAP) from the Research and Development Service of the Department of Veterans Affairs (A.R., R.S.). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: galvanv@uthscsa.edu
Introduction
Alzheimer’s disease (AD), the most common neurodegener-
ative disorder in the elderly[8], is currently without effective
treatment. The accumulation of soluble oligomeric forms of the
amyloid-b peptide (Ab), derived from proteolytic processing of
the amyloid precursor protein (APP), is a major cause of
neurotoxicity in AD[8]. The greatest known risk factor for AD is
increasing age. PDAPP [also known as hAPP(J20)] mice are a
well-defined mouse model of AD[9,10]. PDAPP mice accumu-
late soluble and deposited Ab and develop AD-like synaptic
deficits as well as cognitive impairment and hippocampal
atrophy[9,10,11].
The target of rapamycin (TOR) pathway is a major signaling
hub that integrates nutrient/growth factor availability with cell
metabolism[12] through two distinct complexes, mTORC1 and
mTORC2[13]. mTORC1 functions as a nutrient/energy/redox
sensor and controls protein synthesis. In addition, mTORC1
inhibits autophagy when nutrients and energy are plentiful
through the phosphorylation of Unc51-like kinase 1 (ULK1) and
mAtg13, the mammalian homologs of the yeast kinase Atg1 and
Atg13 respectively, which are essential for the formation of pre-
autophagosomal structures [14]. Phosphorylation of ULK1 and
mAtg13 inhibits ULK1 activity.
mTOR also regulates autophagy through mTORC2. Active
mTORC2 phosphorylates and activates Akt and PKC[15,16].
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2. Since Akt positively regulates mTORC1, phosphorylation of Akt
by mTORC2 stimulates mTORC1 function, inhibiting autoph-
agy. In addition, phosphorylated Akt blocks the activation of
FOXO3, a member of the FOXO family of transcription factors
that have a key role in lifespan extension in invertebrates. Among
many targets, FOXO factors regulate expression of autophagy-
related genes[17]. Thus mTORC2 activity indirectly inhibits
autophagy through inactivation of FOXO3.
Autophagy is a major degradation pathway for organelles and
aggregated proteins[18] such as those that cause multiple
neurodegenerative diseases including AD. It has been reported
that autophagy is activated in AD brains[19]. While excessive
autophagic activity can lead to cell death, increased autophagy has
been shown to facilitate the clearance of aggregation-prone
proteins such as Ab[20,21,22], pathological prion protein[23,24],
and a-synuclein[25], and to promote neuronal survival in a variety
of neurodegenerative disease models. Supporting the notion that
autophagy may have a protective role in AD, deletion of the beclin
1 gene in PDAPP mice impaired autophagy and resulted in large
increases in Ab levels and accelerated Ab deposition[26]. On the
other hand, the endosomal-lysosomal system is a major site of Ab
production[27,28] and it was recently demonstated that Ab is
generated during macroautophagy both in vitro and in vivo[19].
The role of the mTOR pathway and of autophagy in AD is thus
still unclear.
A recent report showed that long-term treatment with
rapamycin, an inhibitor of the mTOR pathway[5], or ablation
of the mTOR target S6K1[6] extends lifespan in mice, possibly by
delaying aging. Whether inhibition of the mTOR pathway would
delay or prevent age-associated disease such as AD remained to be
determined. Here we show that long-term mTOR inhibition by
rapamycin inhibited mTOR in brain, prevented AD-like cognitive
deficits (Fig. 1) and lowered levels of Ab42 (Fig. 2) in the PDAPP
transgenic mouse model. These data indicate that inhibition of the
mTOR pathway by long-term rapamycin treatment can reduce
Ab42 levels in vivo and block or delay AD in mice. As expected
from the inhibition of mTOR, autophagy was strongly activated in
hippocampus of rapamycin-treated mice. Activation of autophagy
was prominent in transgenic, but not in non-transgenic, PDAPP
mice (Fig. 3), suggesting that the reduction in Ab and the
improvement in cognitive function may be due in part to increased
autophagy in neurons, possibly as a response to high levels of Ab in
transgenic mice. Consistent with this hypothesis, inhibition of
mTOR by rapamycin had no effect on endogenous mouse Ab
levels in non-transgenic brains, in which the autophagic response
was not activated. Thus, our data suggest that inhibition of mTOR
by rapamycin, an intervention that extends lifespan in mice, can
lower Ab levels and slow or block AD progression in a transgenic
mouse model of the disease, possibly by stimulating autophagy.
Rapamycin, already used in clinical settings, may be a potentially
effective therapeutic agent for the treatment of AD.
Results and Discussion
A recent report showed that microencapsulated rapamycin, an
inhibitor of the mTOR pathway[5], or genetic ablation of the
mTOR target S6K1[6] extends lifespan in mice, possibly by
retarding aging. Whether rapamycin would prevent or delay age-
associated disease such as AD was unknown. To answer this question,
we fed a rapamycin-supplemented diet identical to the diet that
extended lifespan in mice[5] or control chow to groups of PDAPP
mice and littermate non-transgenic controls for 13 weeks starting at 4
months of age. At 4 mo, hAPP(J20) PDAPP mice
[9,10,11,29,30,31,32] show high Ab levels and synaptic dysfunc-
tion[11,32], but no Ab plaques or spatial memory impairments. At
the end of treatment (7 mo), learning and memory were tested using
the Morris water maze [11,31,33,34]. Consistent with our and others’
previous observations [11,29,31,32,35], we observed significant
deficits in learning and memory in control-fed transgenic PDAPP
animals (Figure 1a and b). Rapamycin-fed transgenic PDAPP mice,
however, showed improved learning (Figure 1a) and memory
(Figure 1b), with improved performances on the last day of training
and retention of the former location of the escape platform restored to
levels indistinguishable from those of non-transgenic littermates
(Figure 1b). Although no significant interactions were observed
during training between day number and genotype, nor between day
number and treatment for control-fed animals, a significant
interaction between treatment and training day was observed, with
increasingly lower latencies (thus increasingly improved performance)
for rapamycin-fed PDAPP transgenic animals as training progressed
(Figure 1a). This observation suggests that incremental learning
during the acquisition phase may be accelerated or improved in
rapamycin-treated PDAPP transgenic mice. A trend to improved
performance during both the acquisition and testing phases was
observed in rapamycin-treated non-transgenic animals, but these
differences did not reach statistical significance. Thus, our data
indicate that rapamycin treatment can ameliorate learning deficits
and abolish memory impairments in PDAPP mice. No significant
differences in thigmotaxis and floating, measures of anxiety and
helplessness respectively (Figure 1c and d) nor in visual acuity
(Figure 1a, inset) were found among groups, suggesting that
improved performance in rapamycin-treated PDAPP mice is a result
of effects on cognitive processes but not to effects related to non-
cognitive components of behaviour, nor to differences in visual
ability.
To determine the mechanism by which rapamycin treatment
abolishes AD-like cognitive deficits in transgenic PDAPP mice, we
examined (a) mTOR activity and (b) the proteolytic processes that
generate of Ab in PDAPP mouse brains. Phosphorylation of the
mTOR target p70S6K[6] was reduced in brains of both
rapamycin-treated PDAPP mice and non-transgenic littermates,
indicating that mTOR activity was inhibited (Fig. 2a–c)[36,37] in
brains of both genotypes. Levels of Ab42, but not of Ab40, were
reduced in rapamycin-treated transgenic PDAPP mice
(Figure 2d). The reduction in Ab42 in PDAPP brains did not
likely arise from changes in production, since the abundance of
C99, the C-terminal product of b-secretase cleavage of APP, as
well the levels of expression of hAPP were unchanged by
rapamycin (Fig. 2f–h). An increase in a-secretase cleavage could
not explain the reduction in Ab42 either, since increased a-
cleavage would result in increased C83 (Fig. 2f and i). In
addition, the reduction in Ab42 did not likely arise from increased
degradation, since levels of the two major Ab-degrading activities,
neprilysin (NEP)[35], and insulin-degrading enzyme (IDE)[38]
were unchanged in brains of control- and rapamycin-fed PDAPP
mice as well (Fig. 2f, j–k). Ab deposition was not determined
since no Ab plaques are detectable in PDAPP mice at 7 mo. To
determine whether levels of endogenous mouse Ab (mAb) would
be affected by rapamycin treatment we measured mAb42 and
mAb40 in brains of control- and rapamycin-treated non-transgenic
littermates using specific ELISAs. Levels of mouse Ab40 were
unchanged by rapamycin treatment (Fig. 2e). Levels of mouse
Ab42 were below the limits of detection for the ELISA (please see
Methods).
Autophagy is a key pathway for the clearance of aggregation-
prone proteins and may have a protective role in proteinopa-
thies[20,39]. Inhibition of the mTOR pathway by rapamycin
activates autophagy[40]. Moreover, rapamycin-induced autopha-
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3. gy has been implicated in the regulation of amyloid accumulation
in vivo[26] and in the clearance of huntingtin[21,40] and a-
synuclein[41]. The role of autophagy in AD, however, is not
clear[19,27]. The induction of autophagy is associated with
increased levels of microtubule-associated-protein-light-chain-3
(LC3)-II, the lipidated form of LC3[42], with respect to levels of
a control protein such as b-actin or b-tubulin[43]. To determine
whether rapamycin treatment affected autophagy in PDAPP
brains, we examined LC3-II and b-actin in hippocampus of
control- and rapamycin-treated PDAPP mice. LC3-II is created
during autophagosome formation and is subsequently degraded as
autophagosomes mature into autolysosomes. Lysosomal turnover
of LC3-II, commonly termed autophagic flux, is the standard
biochemical measurement for autophagy[43]. During autophagy,
LC3-II on the cytosolic side of autophagosomal membranes is
delipidated to LC3-I and is also degraded intraluminally by
lysosomal hydrolases[43,44]. Thus, decreased LC3-II levels may
be observed as a consequence of robust induction of autophagic
flux[43,44]. In agreement with the expected induction of
autophagy by rapamycin-mediated inhibition of mTOR, LC3-
II/b-actin ratios in hippocampi of rapamycin-treated PDAPP
mice were significantly decreased (Fig. 3a–b). In contrast, no
Figure 1. Rapamycin abrogates memory deficits in PDAPP hAPP(J20) mice. a, Rapamycin improves learning in PDAPP mice. While
learning in both transgenic groups was impaired with respect to wild-type littermates’ [**, P,0.001 for both comparisons, Bonferroni’s post hoc test
applied to a significant effect of genotype and treatment, F(3,120) = 29.46, P,0.0001, repeated measures two-way ANOVA], performance of
rapamycin-fed PDAPP mice was improved with respect to the control-fed transgenic group only in the last day of training (#
P = 0.036 for the
comparison of performance between transgenic groups, Student’s t test), indicating improved learning of rapamycin-fed PDAPP mice at day 4. No
significant interaction was observed between day number and genotype (P = 0.96), indicating that genotype had roughly the same effect at all times
during training. Although no significant interaction was observed between day number and treatment for control-treated animals (P = 0.91), a
significant interaction was observed between day number and treatment for rapamycin-treated groups. The effect of rapamycin treatment became
more pronounced as training progressed, as indicated by the slopes for the learning curves (m = 25.14 for rapamycin-treated as compared to
m = 23.58 for control-treated PDAPP transgenic mice; m = 24 for rapamycin-treated as compared to m = 22.95 for control-treated non-transgenic
mice). A trend to improved learning was observed in rapamycin-treated non-Tg mice, but this difference was not significant. Overall learning was
effective in all groups [F(3,120) = 10.29, P,0.0001, repeated measures two-way ANOVA]. Inset, learning was effective in all experimental groups
during cued training. b, Rapamycin restores spatial memory in PDAPP mice. While retention in control-fed PDAPP mice was impaired with
respect to all other groups, as previously described[11,31,35,50] [P values are indicated, Tukey’s multiple comparisons test applied to a significant
effect of genotype (P,0.0001) in one-way ANOVA], memory in rapamycin-fed PDAPP mice was indistinguishable from that of control- or rapamycin-
fed non-Tg groups. A trend to improved retention was observed in rapamycin-treated non-Tg mice, but this difference did not reach statistical
significance. c and d, Rapamycin treatment does not affect non-cognitive components of behavior. c, Although transgenic groups spent
more time engaged in thigmotactic swim, as described[31] (** P,0.001, Bonferroni’s post hoc test applied to a significant effect of genotype
[F(3,440) = 15.04, P,0.0001, two-way ANOVA], no significant difference in percent time spent in thigmotactic swim was observed between transgenic
groups. d, No significant difference in floating was observed between groups. Data are mean 6 SEM.
doi:10.1371/journal.pone.0009979.g001
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4. differences in LC3-II/b-actin ratios were observed between
control- and rapamycin-treated non-transgenic littermates
(Fig. 3a–b), suggesting that rapamycin may induce autophagy
as a response to high Ab levels in hippocampi of transgenic
PDAPP mice. During autophagy, LC3 redistributes to autophago-
somes, which can be visualized as puncta in individual
cells[19,43,44]. To determine whether the decreased LC3-II/b-
actin ratios in hippocampi of rapamycin-treated PDAPP mice
resulted from the induction of autophagic flux, we examined LC3
distribution, as well as levels of p62SQSTM, an ubiquitin-binding
scaffold protein that is specifically degraded by autophagy[22,45],
in hippocampi of control- and rapamycin-treated PDAPP mice.
LC3-immunoreactive puncta were increased in the projections of
hippocampal neurons of rapamycin-treated PDAPP mice (Fig. 3c–
d), suggesting that LC3 was redistributed to a vesicle-like
compartment. Consistent with this observation, levels of the
autophagosomal substrate p62SQSTM were significantly de-
creased in hippocampi of rapamycin-treated PDAPP mice
(Figure 3f–g). Phosphorylation of p70 was significantly reduced
in hippocampi of both PDAPP transgenic and non-transgenic
littermate controls, indicating that mTOR activity was inhibited
(Fig. 3h–i). Taken together, our results suggest that autophagy is
induced by rapamycin-mediated mTOR inhibition specifically as
a response to high Ab levels in hippocampi of rapamycin-treated
PDAPP mice.
The data presented here are, to our knowledge, the first to show
that inhibition of mTOR by rapamycin decreased Ab42 levels
(Fig. 2) and rescued cognitive function (Fig. 1) in a mouse model
of AD. Our data suggest that the reduction in Ab42 levels and the
improvement in cognitive function in rapamycin-treated PDAPP
mice may be a consequence of the induction of autophagy in
hippocampus (Fig. 3) by high levels of Ab in PDAPP transgenic
brains. Consistent with a key role for high levels of Ab in the
activation of autophagy when mTOR activity is reduced,
rapamycin did not induce autophagy in brains of rapamycin-
treated non-transgenic mice, in which levels of endogenous Ab are
much lower than those in PDAPP transgenic brains. In addition,
rapamycin treatment did not induce autophagy and did not affect
levels of endogenous Ab in non-transgenic mice, suggesting that
autophagy may have a key role in reducing Ab42 in transgenic
PDAPP brains. Rapamycin was administered to PDAPP mice at a
dose that was previously shown to extend lifespan in mice[5]. Our
observations are thus consistent with a recent report that showed
that the life-extending effect of TOR inhibition in C. elegans
requires autophagy[46]. It is possible that the activation of
autophagy as a response to Ab accumulation is reduced with
increasing age[47,48]. This may be a consequence of inactivation
of DAF family member FOXO factors by mTOR signaling during
aging[13]. Prolonged rapamycin treatment may thus release
mTOR-mediated inhibition of autophagy and allow for the
reduction of Ab levels through this clearance mechanism in
transgenic PDAPP brains. Although rapamycin treatment did not
activate autophagy nor reduce endogenous mouse Ab levels, it
inhibited mTOR function in non-transgenic littermate brains, and
this group showed trends to improved learning and retention.
Although the differences in performance between control-fed and
Figure 2. Rapamycin inhibits mTOR and decreases Ab42 levels in brains of PDAPP mice. a, b and f, representative immunoblots of whole
brain lysates from control- and rapamycin-treated PDAPP transgenic and non-transgenic littermate mice; c, g–k, quantitative analyses of protein or
phosphoprotein levels. a–c, Levels of phosphorylated (activated) p70 were decreased in brains of rapamycin-treated non-transgenic (a) and
transgenic PDAPP (b) mice (c, **, P = 0.006 and *, P = 0.01 respectively). d, rapamycin did not alter Ab40 levels but significantly decreased soluble Ab42
levels in the brains of transgenic PDAPP mice *, P = 0.02. Homogenates were measured at 100 mg brain tissue/ml. e, rapamycin did not alter levels of
endogenous mouse Ab40 levels in brains of non-transgenic mice. Ab42 levels were below the detection limit of the ELISA (not shown). f,
representative immunoblots of PDAPP mouse brain extracts. g–k, Quantitative analyses of APP, C99 and C83, NEP and IDE immunoreactivity in
lysates of brains from control- and rapamycin-treated PDAPP mice. Data were normalized to b-actin levels. Student’s t test was used to determine
significance of differences between means. Data are means 6 SEM.
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5. Figure 3. Rapamycin increases autophagy in brains of PDAPP mice. a, f and h, representative immunoblots of hippocampal lysates from
control- and rapamycin-treated transgenic PDAPP mice and non-transgenic littermate controls. b, g and i, quantitative analyses. a and b, LC3-II
levels are decreased in hippocampi of rapamycin-treated transgenic PDAPP mice (*, P = 0.0009), but not in hippocampi of rapamycin-treated non-
transgenic littermates. c and d, representative epifluorescent (c, 2006) and higher-magnification confocal (d, 6006) images of hippocampal CA1 (e,
green box, region of epifluorescent images; blue box, region of confocal images) in control- and rapamycin-fed transgenic PDAPP mice stained with
an anti-LC3 antibody. An increase in LC3-immunoreactive puncta was observed in CA1 projections of transgenic PDAPP mice following rapamycin
administration. f and g, levels of the autophagic substrate p62SQSTM are decreased (*, P = 0.0015) in hippocampi of rapamycin-treated PDAPP
transgenic mice. f, representative Western blots; g, quantitative analyses of p62SQSTM levels. h and i, Levels of phosphorylated (activated) p70 were
decreased in brains of rapamycin-treated PDAPP and non-transgenic mice (*, P = 0.001 and P = 0.04 respectively). Significance of differences between
group means were determined using two-tailed unpaired Student’s t test. Data are means 6 SEM.
doi:10.1371/journal.pone.0009979.g003
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6. rapamycin-fed non-transgenic groups were not significant, they
may suggest that changes in pathways different from autophagy
(such as effects on the regulation of protein synthesis) as a result of
long-term mTOR inhibition may have a positive effect on learning
and memory. Reducing Ab levels abolishes cognitive impairments
in a variety of models[49]. We cannot rule out, however, mTOR-
dependent effects on cognition that may be additive to the benefit
of reduced Ab in our model system.
In summary, our data suggest that inhibition of mTOR by
rapamycin[5], an intervention that extends lifespan in mice[5,6], can
slow or block AD progression in a transgenic mouse model of the
disease. Rapamycin, already used in clinical settings, may thus be a
potentially effective therapy for the prevention or treatment of AD.
Methods
Mice
The derivation and characterization of PDAPP [hAPP(J20)]
mice has been described elsewhere[9,10,29]. PDAPP mice were
maintained by heterozygous crosses with C57BL/6J mice (Jackson
Laboratories, Bar Harbor, ME). PDAPP mice were heterozygous
with respect to the transgene. Non-transgenic littermates were
used as controls. Rapamycin administration and behavioral
experiments involving PDAPP mice were conducted at the Buck
Institute for Age Research, Novato, CA. Experimental groups
were: control-fed non-Tg, n = 10; rapamycin-fed non-Tg, n = 10;
control-fed Tg, n = 12; rapamycin-fed Tg, n = 12, all animals were
males and 7 month-old at the time of testing. Rapamycin was
administered for 13 weeks starting at 4 months of age.
Rapamycin treatment
Mice were fed chow containing either microencapsulated
rapamycin at 2.24 mg/kg or a control diet as described by Harrison
et al.[5]. Rapamycin was used at 14 mg per kg food (verified by
HPLC). On the assumption that the average mouse weighs 30 gm
and consumes 5 gm of food/day, this dose supplied 2.24 mg
rapamycin per kg body weight/day[5]. All mice were given ad libitum
access to rapamycin or control food and water for the duration of the
experiment. Body weights and food intake were measured weekly.
Food consumption remained constant for both control- and
rapamycin-fed groups during treatment (no significant effect of week
# on food consumption by two-way ANOVA, P =0.108). Food
consumption was higher for rapamycin-fed animals (by an average of
2.1260.22 g/mouse/week at all times during the experiment
(P,0.001, two-way ANOVA). This may be a result of the inhibition
of the mTOR pathway, which is expected to mimic the unfed state by
decreasing mTOR activity. Littermates (transgenic and non-
transgenic mice) were housed together, thus we could not distinguish
effects of genotype on food consumption. In spite of the differences in
food consumption, overall body weight of control- and rapamycin-fed
groups was not significantly different (25.5960.43 to 26.8960.44 for
control-fed and 26.7760.48 to 28.1160.53 for rapamycin-fed
animals) although body weight increased moderately for both groups
during the 13 week treatment, possibly as a result of the change in
base chow composition (increases were 5% and 11% for rapamycin-
fed transgenic and non-transgenic groups respectively; 10 and 15%
for control-fed transgenic and non-transgenic groups respectively).
The relatively higher increase in body weight for non-transgenic
animals is not unexpected, since non-transgenic animals tend to be
slightly (1–3 g) heavier than PDAPP transgenic mice.
Behavioral testing
The Morris water maze (MWM)[11,31,33] was used to test
spatial memory. All animals showed no deficiencies in swimming
abilities, directional swimming or climbing onto a cued platform
during pre-training and had no sensorimotor deficits as deter-
mined with a battery of neurobehavioral tasks performed prior to
testing. All groups were assessed for swimming ability 2 days
before testing. The procedure described by Morris et al.[33] was
followed as described[11,31]. Briefly, transgenic and non-trans-
genic PDAPP mice were given a series of 6 trials, 1 hour apart in a
light-colored tank filled with opaque water whitened by the
addition of non-toxic paint at a temperature of 24.061.0uC. In the
visible portion of the protocol, animals were trained to find a
12612-cm submerged platform (1 cm below water surface)
marked with a colored pole that served as a landmark placed in
different quadrants of the pool. The animals were released at
different locations in each 60-second trial. If mice did not find the
platform in 60 seconds, they were gently guided to it. After
remaining on the platform for 20 seconds, the animals were
removed and placed in a dry cage under a warm heating lamp.
Twenty minutes later, each animal was given a second trial using a
different release position. This process was repeated a total of 6
times for each mouse, with each trial ,20 minutes apart. In the
non-cued part of the protocol, the water tank was surrounded by
opaque dark panels with geometric designs at approximately
30 cm from the edge of the pool, to serve as distal cues. The
animals were trained to find the platform with 6 swims/day for 4
days following the same procedure described above. At the end of
training, a 30-second probe trial was administered in which the
platform was removed from the pool. The number of times that
each animal crossed the previous platform location was deter-
mined as a measure of platform location retention. During the
course of testing, animals were monitored daily, and their weights
were recorded weekly. Performance in all tasks was recorded by a
computer-based video tracking system (Water2020, HVS Image,
U.K). Data were analyzed offline by using HVS Image and
processed with Microsoft Excel.
Western blotting and Ab determinations
Mice were euthanized by isoflurane overdose followed by
cervical dislocation. Hemibrains were flash frozen. One hemibrain
was homogenized in liquid N2 while the other was used in
immunohistochemical determinations (5–6 per group) and for
hippocampal dissections (5–6 per group). Half brains were
microdissected to isolate the hippocampus by peeling away the
cortex from the underlying hippocampus and releasing the
hippocampus from the surrounding tissue, in particular the
fimbria, by using fine surgical tweezers (Fine Science Tools) and
then lifting toward the midline. The hippocampus separates easily
but does include some adjacent white matter, which is then
carefully tweezed off. We also obtained hippocampal tissue from at
least 12610 mm unfixed frozen sections mounted on glass slides.
All but the hippocampal area was removed using a scalpel under a
SZ60 Olympus dissecting microscope. The hippocampal tissue
itself was removed by beading 10 ml of RIPA lysis buffer (25 mM
Tris-HCl, pH 7.6; 150 mM NaCl; 1% NP40; 1% sodium
deoxycholate; 0.1% sodium dodecyl sulfate) on it and then
pipetting it up. For Western blot analyses, proteins from soluble
fractions of brain LN2 homogenates and from hippocampal
dissections were resolved by SDS/PAGE (Invitrogen, Temecula,
CA) under reducing conditions and transferred to a PVDF
membrane, which was incubated in a 5% solution of non-fat milk
or in 5% BSA for 1 hour at 20uC. After overnight incubation at
4uC with primary antibodies, the blots were washed in TBS-
Tween 20 (TBS-T) (0.02% Tween 20, 100 mM Tris pH 7.5;
150 nM NaCl) for 20 minutes and incubated at room temperature
with appropiate secondary antibodies. The blots were then washed
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7. 3 times for 20 minutes each in TBS-T and then incubated for
5 min with Super Signal (Pierce, Rockford, IL), washed again and
exposed to film or imaged with a Typhoon 9200 variable mode
imager (GE Healthcare, NJ). Human Ab40 and Ab42 levels, as well
as endogenous mouse Ab40 levels were measured in guanidine
brain homogenates using specific sandwich ELISA assays
(Invitrogen, Carlsbad, CA) as described[11].
Antibodies
Antibodies used were: anti-IDE (Abcam, ab32216); anti-NEP
(R&D AF1126); anti-APP (CT15 (REF); anti-phospho-p70 (Cell
Signaling, #9206); anti-b-actin (Sigma, A3853); anti-LC3 (Novus
Biologicals, NB100-2331); anti-p62 (Progen, GP62-C).
Immunohistochemistry
Ten-micrometer coronal cryosections from snap-frozen brains
were post-fixed in 4% paraformaldehyde and stained with LC3-
specific antibodies (10 mg/ml, Nous, Littleton, CO) followed by
AlexaFluor488-conjugated donkey anti-rabbit IgG (1:500, Molec-
ular Probes, Invitrogen, CA), and imaged with a epifluorescence
microscope (Nikon Eclipse E800 with a FITC cube) or with a laser
scanning confocal microscope (Zeiss LSM 510) using a 488 Argon
laser and a 505 long-pass filter. Images were obtained using 206
and 606 objectives. Z-stacks of confocal images were processed
using LSM Viewer software (Zeiss). All images were collected in
the stratum radiatum of the hippocampus immediately beneath
the CA1 layer at Bregma ,22.18. The MBL Mouse Brain Atlas
was used for reference.
Statistical analyses
Statistical analyses were performed using GraphPad Prism
(GraphPad, San Diego, CA) and StatView. In two-variable
experiments, two-way ANOVA followed by Bonferroni’s post-hoc
tests were used to evaluate the significance of differences between
group means. When analyzing one-variable experiments with
more than 2 groups, significance of differences among means was
evaluated using one-way ANOVA followed by Tukey’s post-hoc
test. Evaluation of differences between two groups was evaluated
using Student’s t test. Values of P,0.05 were considered
significant.
Acknowledgments
We thank Ms. Katrine Krueger (Barshop Institute, University of Texas
Health Science Center at San Antonio) and Molly Susag (Buck Institute for
Age Research) for excellent administrative support.
Author Contributions
Conceived and designed the experiments: VG. Performed the experiments:
PS NP MJH. Analyzed the data: JD AR RS VG. Contributed reagents/
materials/analysis tools: RS. Wrote the paper: VG. Principal investigator
of the study: VG. Responsible for the project design: VG. Supervised
technical personnel: VG. Interpretation of results: VG. Preparation of the
manuscript: VG AR RS. Performance of behavioral experiments: VG, PS.
Conducted immunohistochemical experiments: PS. Conducted all bio-
chemical experiments: NP MJH. Key review of the manuscript: MJH JD.
Assistance in the analysis of biochemical data: MJH. Interpretation of the
autophagy data: JD. General colony maintenance and monitoring: OG.
Performed euthanasia, tissues dissection and preparation of PDAPP mice:
OG. Provided resources and partial support during treatment and testing
of PDAPP mice at the Buck Institute: DEB. Prepared the rapamycin-
containing diet and tested it for rapamycin levels: RS.
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